This invention relates generally to vaccines for poultry and other birds and, more particularly, to vaccines for protecting poultry and other birds against infection by avian pathogenic gram-negative bacteria.
Avian pathogenic E. coli (APEC) strains cause a number of related diseases in poultry and other birds, including air sacculitis, cellulitis, colibacillosis, coligranuloma, colisepticemia, Hjarre""s disease, omphalitis, peritonitis, salpingitis, synovitis (Gross, W. B. in Diseases of Poultry, Calnek et al., eds., Iowa State University Press, p. 138-144, 1991; Messier et al., Avian Diseases 37:839-844, 1993). These diseases and other diseases caused by gram-negative avian pathogens can lead to increased rates of feed conversion carcass condemnation, or death of the animal, resulting in millions of dollars lost to the poultry industry each year. Norton, R. A. Broiler Industry, February 1998, pp. 28-32.
Contamination of poultry products by Salmonella is a significant source of Salmonella infection in humans, which causes gastroenteritis, and thus is a major public health concern. Oral administration of live cells from S. typhimurium strains having attenuating deletions in the cya and crp genes has been shown to provide excellent protection against wild-type Salmonella challenge in chickens (Hassan et al., Res. Microbiol. 141:839-850, 1990; Hassan et al., Infect. Immun. 62:5519-5527, 1994) and systems for stable expression of heterologous antigens in such strains have been developed (Hone et al., Microb. Pathog. 5:407-418, 1988; Strugnell, et al., Gene 88:57-63, 1990; Galan et al., Gene 94:29-35; 1990; Nakayama et al., Bio/Technology 6:693-697, 1995).
APEC strains are represented primarily by only a few serotypes, O1, O2, O35 and O78 (Cloud et al., Avian Dis. 29:1084-1093, 1985, Glantz et al., Avian Dis. 6:322-328, 1962; Gross, supra), while Salmonella serotypes most prevalent in poultry are in the B, C and D groups. O-serotypes of gram-negative bacteria such as E. coli and Salmonella are determined at the molecular level by the so-called O-antigen structure, also termed O-specific chain or O-polysaccharide (O-PS), which generally is comprised of varying lengths of polymerized identical sugar units anchored in the bacterial outer membrane as the outermost component of lipopolysaccharide (LPS) molecules (Helander et al., in Molecular Biology and Biotechnology: A Comprehensive Desk Reference, R. A. Meyers, ed., VCH Publishers, Inc., 1995).
Specific antibody against LPS O-antigen has been shown to be protective in mammalian models of extraintestinal E. coli infections in humans (Cryz et al., Vaccine 13;449-453, 1995; Pluschke et al., Infect. Immun. 49:365-370, 1985) and LPS O-antigen has been recognized as a protective antigen for other gram negative pathogens (Ding et al., J. Med. Microb. 31:95-102, 1990; Michetti et al., Infect. Immun. 60:1786-1792, 1992; Robbins et al., Clin. Infect Dis. 15:346-361, 1992). In addition, several research groups have reported using attenuated Salmonella and Salmonella-E. coli hybrids as vaccine delivery vehicles for O-antigens of several human pathogens, including Shigella sonnei, Vibrio cholerae, and Pseudomonas aeruginosa. (13lack et al., J. Infect. Dis. 155:1260-1265, 1987; Formal et al., Infect. Immun. 34:746-750, 1981; Pier et al., Infect. Immun. 63:2818-2825, 1995); (Morona et al., U.S. Pat. No. 5,110,588). However, until the work described herein, immunization of poultry with live, attenuated Salmonella expressing an APEC O-antigen had not been reported.
LPS O-antigen made by E. coli and Salmonella bacteria is comprised of lipid A, an R-core oligosaccharide, and the O-specific polysaccharide (O-PS), which are covalently linked in that order. Sugiyama et al., J. Bacteriol. 173:55-58, 1991. In S. typhimurium, synthesis of the R-core moiety is directed by the rfa locus and certain housekeeping genes such as galE, galU, and pgi, while O-PS synthesis is directed by the rfb gene cluster, which encodes enzymes involved in biosynthesis of the monomer sugar unit, and the rfc gene, which encodes the O-antigen polymerase responsible for the polymerization of the sugar unit into a high molecular weight polysaccharide chain. Sugiyama et al., supra.
One group investigating the genes required for synthesis of LPS O-antigen in E. coli O9 introduced a plasmid containing the rfb locus from E. coli O9 into S. typhimurium wild-type and mutant strains with defects in the rfb, rfc, or rfe loci and reported that the wild-type strain containing the plasmid expressed LPS specific for both E. coli O9 and S. typhimurium on the cell surface, while the rfc mutant was expressed only O9-specific LPS. E. coli O-antigen was also synthesized in the S. typhimurium rfb mutant but not in the rfe mutant. Sugiyama et al., supra. This group concluded that gene products of the S. typhimurium rfa and E. coli O9 rfb loci can cooperate to synthesize E. coli O9-antigen on the R-core of S. typhimurium. However, this group did not report whether any of these recombinant S. typhimurium constructs could grow within an animal host or generate a protective host immune response against wild-type E. coli O9 or S. typhimurium. Accordingly, a need exists for a bivalent vaccine to control Salmonella and E. coli infection in poultry. Such a vaccine would simultaneously benefit the public health and reduce the costs of poultry production.
In one embodiment the present invention is directed to a vaccine that protects birds against infection by an avian pathogenic gram-negative (APG-N) microbe. The vaccine comprises live cells of a recombinant Salmonella strain expressing O-antigen of the APG-N microbe due to integration into the Salmonella chromosome of the rfb gene cluster and the rfc gene of the APG-N (hereinafter used interchangeably with APG-N rfb/rfc gene cluster). The recombinant Salmonella strain, which is an attenuated mutant of a virulent Salmonella strain, does not express O-antigen of the virulent Salmonella strain due to a mutation in the Salmonella rfb gene cluster and/or in the Salmonella rfc gene. In a preferred embodiment, the APG-N microbe is an APEC strain and the APG-N rfb/rfc gene cluster is an APEC rfb/rfc gene cluster.
This recombinant Salmonella strain has other features that make it particularly useful as a vaccine in poultry and other birds. First, the vaccine can be formulated for oral administration and oral vaccines are known to stimulate the gut associated lymphoid tissue (GALT), including mucosal, humoral and cellular immune responses. Oral, live vaccines also cost less to produce and are easier to administer in the field than injectable vaccines. Second, the lack of expression of LPS O-antigen specific for the carrier strain avoids any interference that the carrier LPS O-antigen might have on expression of the APG-N O-antigen or its recognition by the vaccine recipient""s immune system. Third, the recombinant Salmonella strain can protect against both APG-N microbes and the parental Salmonella strain because it expresses other cell-surface antigens of the parental Salmonella strain. In addition, for vaccines expressing O-antigen from an APEC strain, use of Salmonella rather than E. coli as the carrier bacteria should provide a more vigorous immune response against the APEC O-antigen because while S. enterica subspecies persist in the spleen and bursa of Fabricius, E. coli does not effectively invade these lymphoid tissues or is quickly killed even if occasionally successful in entering them.
In some embodiments, the recombinant Salmonella strain used in the vaccine also contains a recombinant polynucleotide encoding a desired gene product. A preferred gene product is an antigen from an avian pathogenic gram-positive (APG-P) microbe or from a eukaryotic avian pathogen.
In another embodiment, the invention provides a multivalent vaccine for immunizing birds against at least two avian pathogenic gram-negative (APG-N) microbes which comprises live cells of a recombinant Salmonella strain expressing an O-antigen of each of the APG-N microbes, the recombinant Salmonella strain having an rfb/rfc gene cluster of each of the APG-N microbes integrated into the Salmonella chromosome and having a mutation in the Salmonella rfb gene cluster or in the Salmonella rfc gene which inactivates expression of Salmonella O-antigen, wherein the recombinant Salmonella strain is an attenuated mutant of a virulent Salmonella strain. In a preferred embodiment, one or both of the APG-N microbes is an APEC strain.
In yet another embodiment, the invention provides a multivalent vaccine for immunizing a bird against at least two APG-N microbes which comprises a mixture of live cells of first and second recombinant Salmonella strains, the first recombinant Salmonella strain having an rfb/rfc gene cluster of a first APG-N microbe integrated into the Salmonella chromosome and expressing O-antigen of the first APG-N microbe and the second recombinant Salmonella strain having an rfb/rfc gene cluster of a second APG-N microbe integrated into the Salmonella chromosome and expressing O-antigen of the second APG-N microbe, wherein each of the first and second recombinant Salmonella strains has a mutation in the Salmonella rfb gene cluster or in the Salmonella rfc gene which inactivates expression of Salmonella O-antigen, and wherein each of the first and second recombinant Salmonella strains is an attenuated mutant of a virulent Salmonella strain. In a preferred embodiment, one or both of the recombinant Salmonella strains in the multivalent vaccine express O-antigen of an APEC strain.
The present invention in other embodiments is directed to methods for immunizing birds against infection by APG-N microbes. These methods include a method for immunizing a bird against an APG-N microbe which comprises administering to the bird an immunologically effective amount of a vaccine comprising live cells of a recombinant Salmonella strain expressing O-antigen of the APG-N microbe, the recombinant Salmonella strain having an rfb/rfc gene cluster of the APG-N microbe stably integrated into the Salmonella chromosome and having a mutation in the Salmonella rfb gene cluster or in the Salmonella rfc gene which inactivates expression of Salmonella O-antigen, wherein the recombinant Salmonella strain is an attenuated mutant of a virulent Salmonella strain. The methods also include a method for simultaneously immunizing a bird against more than one APG-N microbes which comprises administering to the bird an immunologically effective amount of a multivalent vaccine as described above. Also included is a method for simultaneously immunizing a bird against an APG-N microbe and the carrier Salmonella species which comprises administering to the bird an immunologically effective amount of any of the vaccines as described above.
In still another embodiment, the invention provides a method of making a vaccine for immunizing a bird against an APG-N microbe strain. The method comprises the steps of selecting a Salmonella strain capable of colonizing the bird, integrating into the Salmonella chromosome an rjb/rfc gene cluster from the APG-N microbe, introducing a mutation into the Salmonella rfb gene cluster and/or into the Salmonella rfc gene, and isolating recombinant Salmonella bacteria which express O-antigen characteristic of the APG-N microbe but which do not express Salmonella O-antigen. In one embodiment the selected Salmonella strain is an attenuated mutant of a virulent Salmonella strain. In another embodiment, the selected Salmonella strain is a virulent Salmonella strain and the method further comprises the step of introducing into the virulent Salmonella strain an attenuating mutation into a Salmonella virulence gene and isolating mutants having attenuated virulence as compared to the virulent Salmonella strain.
Among the several advantages achieved by the present invention, therefore, is the provision of live, recombinant Salmonella vaccines capable of protecting birds and particularly poultry against infection by APEC strains and other APG-N microbes, and methods for making such vaccines, the provision of multivalent vaccines useful for simultaneously protecting birds against infection by two or more APG-N microbes, the provision of methods for immunizing birds against APEC strains and other APG-N microbes, and the provision of a method for immunizing birds against both APEC and Salmonella bacteria.